1. Field of the Invention
The present invention relates generally to a method and apparatus for partitioning a heavier phase from a lighter phase of a centrifugally separated multiphase fluid specimen. More particularly, the present invention is directed to a method and apparatus for separating platelets, lymphocytes and monocytes from granulocytes and erythrocytes in a blood specimen.
2. Description of the Prior Art
The Immunological analysis of blood generally requires the isolation and separation of the lymphocytes for detailed analysis. In general, two distinct methods are known for the separation of lymphocytes from other blood cells. The first of these methods involves buoyant density centrifugation of cells through a particular newtonian fluid. The most commonly used fluid is known as Ficoll-Paque.TM., a water soluble liquid having a specific gravity of 1.077 g/cc, which is marketed by Pharmacia Fine Chemicals AB, Upsala, Sweden. The second of these methods utilizes a non-newtonian, water-insoluble, thixotropic, gel-like substance which establishes a continuous semi-rigid gel-like seal across the interior of a container between the lighter phase containing the lymphocytes and a heavier phase containing the erythrocytes and granulocytes.
The Ficoll-Paque.TM. method contemplates the following general steps:
(1) A predetermined amount of Ficoll-Paque.TM. is dispensed into the bottom of a container suitable for centrifugation, such as a test tube;
(2) A sample of whole or diluted blood is carefully pipetted onto the surface of the Ficoll-Paque.TM.;
(3) The Ficoll-Paque.TM./blood preparation is then centrifuged at about 400 g for about 30 minutes to provide a layered separation of blood constituents into a top lighter phase containing a mononuclear cell layer including the lymphocytes and a bottom heavier phase containing the erythrocytes and granulocytes which pass into and/or through the Ficoll-Paque.TM.;
(4) An upper plasma layer is removed by pipetting, leaving behind a lymphocyte layer on the surface of the Ficoll-Paque.TM..
(5) A clean Pasteur pipette is used to transfer the lymphocyte layer to a clean centrifuge tube.
The Ficoll-Paque.TM. method has several disadvantages. First, very careful technique by the operator of the Ficoll-Paque.TM. method is required. If the initial introduction of the blood sample is performed carelessly, plasma may be deployed below the surface of the Ficoll-Paque.TM. medium causing reduced local specific gravity of the Ficoll-Paque.TM. which is then inadequate to separate lymphocytes and monocytes from other cells.
Second, careful technique is again required to transfer the lymphocyte layer from the surface of the Ficoll-Paque.TM. to a clean centrifuge tube for washing. It is critical to remove all the interface but a minimum amount of Ficoll-Paque.TM. during this transfer step.
Third, centrifugation forces higher than about 400 g cannot be utilized since the Ficoll-Paque.TM. is water soluble and higher centrifugation forces dilution of the Ficoll-Paque.TM. with the blood plasma, thereby resulting in a change in the Ficoll-Paque.TM. specific gravity and a substantial alteration in the separation efficiency.
Fourth, if during centrifugation lighter phases in the blood are carried into the Ficoll-Paque.TM. medium, they cannot thereafter ascend through the medium. This is due to the low forces produced by the required centrifugation forces of about 400 g.
Fifth, the method requires 1 to 2 hours for completion. A more rapid process would be highly desirable.
A more rapid process is provided by methods which utilize a non-newtonian water immiscible, thixotropic, gel-like substance (hereinafter referred to as a "gel-like substance") for establishing a barrier between the lighter phase containing the lymphocytes and a heavier phase containing the erythrocytes and granulocytes. This method is exemplified by U.S. Pat. No. 4,190,535 to Luderer; U.S. Pat. No. 3,852,194 to Zine; U.S. Pat. No. 4,147,628 to Bennett; U.S. Pat. No. 4,350,593 to Kessler; and U.S. Pat. No. 4,153,739 to Kessler. The use of a gel-like substance to establish a barrier layer between the lighter phase and the heavier phase generally provides a more rapid and easier method for separating the lighter phase and heavier phase than the Ficoll-Paque.TM. method. However, the use of the gel-like substance in a blood separation scheme also has disadvantages. The most important disadvantage is that the the lymphocyte layer which lies immediately above the gel-like substance after the centrifugation step tends to become contaminated with granulocytes which lie immediately below the gel-like substance. It would be desirable to provide a method and apparatus for separating a blood sample into a lighter phase containing lymphocytes and monocytes and a heavier phase containing erythrocytes and granulocytes which is rapid and which provides a distinct uncontaminated sample of lymphocytes for further analysis.